The present invention relates to a process for allele discrimination employing primer extension using an exonuclease deficient polymerase to distinguish matched 3xe2x80x2-ends from mismatched 3xe2x80x2-ends of hybridized primer and target oligonucleotides.
Many diseases are known which have a genetic basis in their etiology and result from the occurrence of mutations in gene sequences present in the genomes of different organisms, especially animals, including humans, afflicted with such diseases. Consequently, methods for detecting slight genetic differences, as small as one nucleotide (called single nucleotide polymorphisms or SNPs), between the genome of a healthy individual and that of a person afflicted with a genetic defect can prove highly valuable in elucidating the nature and causes of such condition. More importantly, obtaining valuable information about such conditions is greatly enhanced if a sensitive process is available for determining small genetic defects, such as a difference of one nucleotide at a particular location in the genome.
A genome is composed of different loci which are themselves composed of one or more genes, which genes may contain variations, so-called alleles, for each system. For example, the immunoglobulin superfamily, which includes, inter alia, the T-cell receptor, the immunoglobulin and the HLA (or human leukocyte antigen) systems, is characterized by the presence of large sequence variations (called polymorphisms). Defects in the immune response, which are due to diverse variations in one or more of the gene arrangements of such systems, may result in disease. Conversely, diseases like cystic fibrosis show varying and complex genetic variations in DNA sequence. Genetic variation may therefore be linked to diseases and their symptoms. Identification of the associated alleles, especially differences in those alleles, may be important in determining the risk of a disease associated with genetic markers or in detecting variations in genes that result in some other malady. Further, the delineation of slight genetic differences can be readily utilized for the diagnosis (even treatment) of certain diseases, as well as furthering efforts toward prevention by identifying persons having the greatest risk of a particular disease. The latter is a critical factor in those situations where early treatment is possible and the development of the disease can be retarded.
Several methods for detecting specific nucleotide variations and genetic polymorphisms in nucleic acids are known. For example, some methods comprise amplifying nucleic acid sequences having nucleotide variations, mutations and polymorphisms, with subsequent detection thereof using allele specific oligonucleotide sequences and a dot blot. This process utilizes allele-specific oligonucleotide sequences that have to be very specific for the nucleotide variation to be detected and offers numerous primer sequences for use therein depending on the DNA sequence to be studied. [See, for example, EP-A-237,362]
R. K. Saiki et al., Proc. Natl. Acad. Sci. USA. 86, 6230-6234 (1989) as well as WO 89/11548 both disclose use of immobilized sequence-specific oligonucleotides. WO 89/11547 discloses methods for determining genotypes having different alleles in the HLA-DP loci. This latter method operates by hybridizing nucleic acid samples with a series of probes which are specific for various segments.
U.S. Pat. No. 5,912,148 discloses a polymerase chain reaction (PCR) method as well as an oligonucleotide ligase assay (OLA) procedure for analyzing complex genetic systems in a single reaction vessel (also see other methods cited therein). This method seeks to determine the products of the OLA reaction using various OLA and PCR probes.
U.S. Pat. No. 5,759,771 discloses a method for determining genotypes by comparing the nucleotide sequences of members of a gene system that flank the polymorphic segments of a particular genetic locus. Here, the compared sequences contain conserved sequences used to amplify the strongly conserved segments from different sources. These are then compared as a means of establishing genotype.
U.S. Pat. No. 5,710,028 discloses a method of simultaneous determination of the identity of nucleotide bases at specific locations in nucleic acids of interest but relies on the use of extension blocking agents, commonly dideoxy-nucleoside triphosphates, to prevent extension in cases where there is a particular nucleotide present at a given location within the target sequence (the latter acting as a template). A similar process is used in U.S. Pat. No. 6,013,431.
Nucleic acid sequence analysis has become important in many research, medical, and industrial fields and a host of methods have been described in the literature. Heretofore, many of these approaches have been motivated by the development of various methods for amplifying target nucleic acids, e.g. polymerase chain reaction (PCR) of U.S. Pat. No. 5,137,806, ligation chain reaction (LCR), and the like, as well as rolling circle amplification (RCA) (See, for example, U.S. Pat. No. 5,854,033; Lizardi et al, Nature Genetics, 19, 225-232 (1998). Such amplification techniques are certainly useful as the basis for developing sensitive and specific diagnostic assays but in some cases these methods may be fairly complex and involved, especially when the system to be analyzed is a complex one, such as a complex genetic system, for example, the highly variable cystic fibrosis locus. Because it may be difficult to identify the amplified product in such systems, post-amplification manipulations may often be necessary, especially in cases other than RCA. One approach used to avoid these problems is that of the oligonucleotide ligation assay (OLA). [U.S. Pat. No. 4,883,750] Here, oligonucleotides are prepared that are complementary to adjacent regions of a target sequence and are capable of hybridizing to the target so that they lie end-to-end and can be ligated when no mismatches occur at or near the contiguous ends. Whenever mismatches occur, ligation is precluded. The result is a set of oligonucleotide pairs that are perfect complements of all the allelic variants of interest at a given locus. By carefully selecting the labeling method, a wide range of alleles can be specifically identified in a single assay. However, such assays can be complicated. [Nickerson et al., Proc. Natl. Acad. Sci. USA 87:8923-8927 (1990)]
Other methods for allele discrimination have relied on template dependent ligation of two adjacent short oligonucleotides. One such oligonucleotide consists of a reverse polarity oligonucleotide containing a primer for RCA and a short target specific sequence terminating at an allele- specific 3xe2x80x2-end residue. A second oligonucleotide is immobilized on a glass slide and anneals next to the target specific oligonucleotide sequence. Template dependent and allele-specific ligation anchors the RCA platform to the slides. Following RCA, products are detected by standard fluorescent and immunochemical techniques. The use of allele specific primers annealing to different circles allows simultaneous detection of various alleles (called multiplexing). Such methods rely on a ligation step as the allele discrimination event. (see Lizardi et al, supra)
A different method employs RCA using padlock probes to detect mutations in cytological samples. However, padlock probes are not always advantageous due to steric hindrance and topological constraints on DNA targets. Such procedures also rely on a ligation step. [see: Nilsson et al, Padlock Probes: Circularizing Oligonucleotides for Localized DNA Detection, Science, 265, 2085-2088 (1994)]
One approach to simplifying these procedures is to eliminate some of the steps, thereby simplifying and speeding the overall procedure. For example, such procedures have the disadvantage of relying on DNA ligation as the allele discrimination step.
The method according to the present invention overcomes these problems while having the overall advantage of being highly efficient and sensitive to single copy genes as well as being sensitive to single mutations (i.e., SNPs). More specifically, advantages include the fact that new mutations can be detected directly and can then be investigated in more detail for functionality (as opposed to mere serological testing of mutated polypeptides and polynucleotides). In addition, the method is simple and thus can be made widely available for use, it can readily be automated for large scale assays, or provided as a kit for manual and spot determinations, or for use in the field, it can be performed either in suspension or using solid supports for ready isolation of products, it is readily amenable to many different methods of detection and is readily adapted to multiplexing so that different alleles, or sets of alleles, can be readily and simultaneously detected. In addition, the methods of the present invention are useful in allele discrimination, detection of SNPs, genotyping, molecular haplotyping and mutation detection, to name but a few of the uses.
The present invention relates to methods of detecting pne or more nucleotides at specific locations within a gene sequence using primer extension by exonuclease deficient polymerase action.
It is therefore one object of the present invention to provide a simple and ready means of genotyping using the ability of a probe to detect mismatches in a target polynucleotide sequence such that the absence of a given mismatch, i.e., a mutated residue, will lead to amplification of a predetermined gene sequence that can be readily detected and wherein the absence of such sequence amplification is a reliable indicator of the presence of a single nucleotide mismatch.
It is a further object of the present invention to provide methods of detecting mismatches at specified nucleotide positions as a means of simple, specific and straightforward allele discrimination as well as for general use in detecting single nucleotide polymorphisms (SNPs), as well as other mutations, and for use in molecular haplotyping.
It is another object of the present invention to provide methods for the amplification of specific gene sequences as a means of detecting mutations in target polynucleotides wherein said target polynucleotides are derived from genomes of animals, especially humans, but also non-humans.
It is still another object of the present invention to provide a means of allele discrimination through rolling circle amplification and tandem DNA sequence formation that is readily amenable to all forms of detection, including by specific probes and labeling agents, especially using fluorescent labels.
It is yet another object of the present invention to provide methods for genotyping through sequence amplification that are readily adaptable to use in suspension, solution or through the use of solid supports for ready isolation of the products of said amplification.
It is yet a still further object of the present invention to provide methods useful in multiple allele discrimination through procedures readily susceptible to known multiplexing techniques, thereby facilitating the simultaneous detection of different alleles in a sample and limited only by the number of fluorophore detectors available and the equipment available for detection.
In another embodiment, the present invention is directed to kits for carrying out the methods of the invention. Preferably, such kits include (a) a plurality of oligonucleotide probes, each oligonucleotide probe of said plurality being capable of hybridizing to one or more target polynucleotides that may or may not possess a mismatch with respect to a terminal residue of the oligonucleotide probes; (b) a sample of an exonuclease deficient DNA polymerase; (c) a plurality of amplification primers, each said primer being capable of hybridizing to an elongated segment of said oligonucleotide probe as well as comprising a primer sequence complementary to a sequence on an amplification target circle (ATC) for use in rolling circle amplification; (d) a sample of amplification target circles (ATC), essentially single stranded DNA circles, comprising a sequence of 10 to 20, even 30, nucleotides in length, which sequence is complementary to a sequence of the amplification primers of part (c) and which ATCs act as templates for rolling circle amplification (RCA); (e) a sample of a DNA polymerase capable of carrying out rounds of rolling circle amplification, such as T7 DNA polymerase; and (f) a means for detecting the products of rolling circle amplification, including, but not limited to, various labeling reagents and address probes and tags.